The sweet potato RbcS gene (IbRbcS1) promoter confers high-level and green tissue-specific expression of the GUS reporter gene in transgenic Arabidopsis.

GENE-40497; No. of pages: 7; 4C: Gene xxx (2015) xxx–xxx

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Research paper

The sweet potato RbcS gene (IbRbcS1) promoter confers high-level and green tissue-specific expression of the GUS reporter gene in transgenic Arabidopsis Noriaki Tanabe, Masahiro Tamoi, Shigeru Shigeoka ⁎ Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

a r t i c l e

i n f o

Article history: Received 25 March 2015 Received in revised form 29 April 2015 Accepted 2 May 2015 Available online xxxx Keywords: Sweet potato RbcS Promoter activity

a b s t r a c t Sweet potato is an important crop because of its high yield and biomass production. We herein investigated the potential of the promoter activity of a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) from sweet potato (Ipomoea batatas) in order to develop the high expression system of exogenous DNA in Arabidopsis. We isolated two different cDNAs (IbRbcS1 and IbRbcS2) encoding RbcS from sweet potato. Their predicted amino acid sequences were well conserved with the mature RbcS protein of other plants. The tissue-specific expression patterns of these two genes revealed that expression of IbRbcS1 was specific to green tissue, whereas that of IbRbcS2 was non-photosynthetic tissues such as roots and tubers. These results suggested that IbRbcS1 was predominantly expressed in the green tissue-specific of sweet potato over IbRbcS2. Therefore, the IbRbcS1 promoter was transformed into Arabidopsis along with β-glucuronidase (GUS) as a reporter gene. GUS staining and semiquantitative RT-PCR showed that the IbRbcS1 promoter conferred the expression of the GUS reporter gene in green tissue-specific and light-inducible manners. Furthermore, qPCR showed that the expression levels of GUS reporter gene in IbRbcS1 pro:GUS were same as those in CaMV 35S pro:GUS plants. These results suggest that the IbRbcS1 promoter is a potentially strong foreign gene expression system for genetic transformation in plants. © 2015 Published by Elsevier B.V.

1. Introduction Because more than 90% of crop biomass is derived from photosynthetic products, enhancement of the photosynthetic capacity of plants has been reported to increase crop yields (Yamori, 2013). Plant transformations have been proposed to increase the inherent yielding capability of plants (Sinclair et al., 2004). In the Calvin cycle, sedoheptulose 1,7bisphosphatase (SBPase), transketolase (TK), and fructose 1,6bisphosphate aldolase (aldolase) are considered to have higher control coefficients on photosynthesis than other Calvin cycle enzymes and, thus, are candidate targets of engineering to improve photosynthetic carbon fixation (Raines, 2011). However, significant improvements in plant yield by engineering carbon assimilation have only been achieved in a few cases (Miyagawa et al., 2001; Yokota and Shigeoka, 2008; Uematsu et al., 2012). We previously reported that transgenic tobacco and lettuce plants overexpressing the fructose-1,6-/sedoheptulose1,7-bisphosphatase (FBP/SBPase) of Synechococcus elongatus PCC7942 (S.7942) in their chloroplasts showed an enhanced CO2 assimilation

Abbreviations: RbcS, small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase; GUS, β-glucuronidase. ⁎ Corresponding author at: Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan. E-mail address: [email protected] (S. Shigeoka).

rate and increased biomass production (Miyagawa et al., 2001; Yabuta et al., 2008; Ichikawa et al., 2010). Promoters are regions of DNA upstream of the coding region that contain specific sequences, which regulate gene transcription. Constitutive promoters such as the Cauliflower mosaic virus 35S (CaMV 35S) are widely used in the genetic engineering of plants. However, since the constitutive expression of foreign proteins in transgenic plants can often have adverse effects, the utilization of an appropriate promoter is important for the transgene expression (Bakhsh et al., 2011). Ribulose-1,5bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the initial carbon dioxide fixation step in the Calvin cycle and also catalyzes the oxygenase reaction leading to the photorespiration (Hartman and Harpel, 1994). In higher plants, Rubisco comprises eight large subunits (RbcL) and eight small subunits (RbcS) (Andersson and Bachlund, 2008). RbcL is encoded by a single gene on the chloroplast genome, while RbcS is encoded by a multigene family in the nuclear genome. The RbcS multigene family consists of 2–22 members, depending on the species (Sasanuma, 2001; Spreitzer, 2003). RbcS polypeptides are formed as precursors that contain a transit peptide, which is removed during transport into chloroplasts. Rubisco is the most abundant protein found in plant leaves and comprises approximately 50–60% of soluble protein (Ellis, 1979). Thus, RbcS promoters and their transit peptides are attractive candidates for the expression of exogenous genes at high levels in green tissue and the targeting of exogenous proteins into chloroplasts.

http://dx.doi.org/10.1016/j.gene.2015.05.006 0378-1119/© 2015 Published by Elsevier B.V.

Please cite this article as: Tanabe, N., et al., The sweet potato RbcS gene (IbRbcS1) promoter confers high-level and green tissue-specific expression of the GUS reporter gene in transgenic Arabidopsis, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.006

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N. Tanabe et al. / Gene xxx (2015) xxx–xxx

Sweet potato (Ipomoea batatas) is one of the most important crops in the world and represents an attractive target for molecular breeding because of its clonal propagation, ease of cultivation, and the high productivity of its storage roots and leaves. However, sweet potato is a genetically hexaploid plant and its genomic sequence has not yet been decoded; therefore, few studies have examined gene promoters from sweet potato. The promoter activity of sweet potato ADP-glucose pyrophosphorylase (IbAGP1) was previously shown to confer the high-level expression of the GUS gene in the leaves and tubers of a transgenic potato (Kim et al., 2009). Furthermore, the promoter activity of sweet potato sporamin was highly active in the tubers of a transgenic potato (Hong et al., 2008). However, information regarding green tissue-specific promoters from sweet potato is limited. In the present study, we cloned two RbcS genes (DDBJ accession numbers LC036584 and LC036585) from the sweet potato genome. We demonstrated the green tissue-specific expression of the IbRbcS1 gene and studied the promoter activity of the 5′-flanking sequences of IbRbcS1 using GUS as a reporter gene in Arabidopsis plants. 2. Materials and methods 2.1. Plant materials and growth conditions Sweet potato (I. batatas L. cv. Kokei 14) plants were grown in soil under 28 °C/23 °C day–night temperatures and 14-h photoperiod (light intensity of 500 μmol m−2 s−1). Arabidopsis (Arabidopsis thaliana)

ecotype Columbia-0 plants were grown on MS medium (Murashige and Skoog, 1962) containing 3% sucrose under 25 °C/23 °C day–night temperatures and 14-h photoperiod (light intensity of 100–150 μmol m−2 s−1). 2.2. Isolation of IbRbcS genes All primers used for the isolation of IbRbcS genes are listed in Supplemental Table S1. Genomic DNA was isolated from the leaves of sweet potato using DNA suisui-VS (Rizo, Tsukuba, Japan). Isolated genomic DNA was treated with RNase A (Thermo scientific, Waltham, MA) before being subjected to PCR. DNA fragments of IbRbcS1 and IbRbcS2 were amplified by PCR using degenerated primers (Firon et al., 2013). The PCR amplification was performed using Gflex polymerase (Takara, Shiga, Japan) with an initial enzyme activation step of 2 min at 95 °C, followed by 35 cycles of 10 s at 98 °C, 15 s at 55 °C, 15 s at 68 °C, and a final extension of 5 min at 68 °C. The PCR products obtained were directly cloned into the pMD20-T vector using the Mighty TA-cloning Reagent set for PrimeSTAR (Takara, Shiga, Japan). Insert-containing clones were then grown and the plasmid DNAs were isolated. Nucleotide sequences were analyzed using a Big-Dye terminator Cycle Sequencing Ready Reaction Kit and ABI 3100-Avant Genetic Analyzer (Applied Biosystems, CA, USA). The 5′- and 3′-flanking regions of IbRbcS genes were isolated using an LA PCR in vitro cloning Kit (Takara, Shiga, Japan). Genomic DNA was digested for 5 h using one of the seven restriction enzymes (EcoRI, XbaI, SalI, HindIII, BamHI, PstI, or Sau3A) and ligated with each restriction

Fig. 1. Nucleotide sequences of (A) IbRbcS1 and (B) IbRbcS2 genes. The non-coding regions including the protein coding regions and 150-bp of the flanking region are shown. Uppercase and lowercase letters indicate the coding region and non-coding region, respectively.



enzyme cassette, respectively. The first PCR reaction was performed using the ligated genomic DNA as a template with a cassette and gene-specific primer. The PCR amplification steps was performed using Gflex polymerase with an initial enzyme-activation step of 2 min at 95 °C, followed by 35 cycles of 10 s at 98 °C, 15 s at 55 °C, 1 min at 68 °C, and a final extension of 5 min at 68 °C. The PCR products were used as a template for second PCR with a cassette and gene-specific primer following the PCR procedure described above. The PCR products were cloned into the pMD20-T vector and sequenced as described above. 2.3. Construction of plant expression vector and generation of transgenic Arabidopsis plants

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site) was amplified with primers containing HindIII and NdeI at the 5′ and 3′ ends of the IbRbcS1 promoter gene, respectively, and cloned into pMD20-T. The promoter fragment was ligated upstream of GUS in the binary vector pRI201-AN GUS (Takara, Shiga, Japan). Arabidopsis was transformed via Agrobacterium tumefaciens-mediated gene transfer. Transformed seeds were selected in the T1 generation on MS plates with 50 μg/mL kanamycin. Seed batches that contained single insert heterozygote seeds and showing a 1/4 death ratio on the selection plates were used to obtain homozygote lines at T2 stage. Transgenic lines designated IbRbcS1 pro:GUS 4-1 and 6-3 at T3 generation, were used in subsequent experiments. 2.4. RT-PCR

All primers used for plasmid construction are listed in Supplemental Table S1. To construct IbRbcS1 pro:GUS chimeric genes, the 5′-flanking region of IbRbcS1 (from −991 to −1, numbered from the ATG initiation

All primers used for the RT-PCR are listed in Supplemental Table S1. Total RNA was extracted from the various tissues of sweet potato using

Fig. 2. Multiple alignments of deduced amino acid sequences of RbcS among Ipomoea batatas RbcS1 and S2, Arabidopsis thaliana (GenBank accession no. P10797), Nicotiana tabacum (P69249), Solanum tuberosum (P26577), Solanum lycopersicum (CAA29402), and Zea mays (NP_001105294). The amino acid residues common to at least four among species with IbRbcS1 or IbRbcS2 are colored gray. The filled arrowhead shows the cleavage site of the transit peptide for targeting to the chloroplast. The open arrowheads show the positions of the intron in IbRbcS1 and IbRbcS2. Sequences were analyzed with GENETYX-MAC Version 17.


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(bp) 1000 900 800 700 600 500 400 300

1000 900 800 700 600 500 400 300

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L

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MA). After extraction with a phenol:chloroform (1:1) solution and ethanol precipitation, 1st strand cDNA was synthesized from 1 μg of total RNA using the PrimeScript 1st strand cDNA Synthesis Kit (Takara, Shiga, Japan). Semi-quantitative RT-PCR was performed using Gflex polymerase and the primers listed in Supplemental Table S1. DNA polymerase was first activated at 94 °C for 2 min, and PCR was carried out for 23 to 26 cycles of 10 s at 98 °C, 15 s at 55 °C, and 15 s at 68 °C, followed by a final extension step for 5 min at 68 °C. 2.5. GUS staining

IbRbcS2

Actin Fig. 3. Expression of IbRbcS1 and IbRbcS2 mRNAs in various tissues of sweet potato. Actin was used as a control for the experiments. Amplified products were electrophoresed on 1.5% (w/ v) agarose gels. L, leaves; S, stems; R, roots; T, tubers; M, 100 bp Ladder DNA marker (Takara).

RNA suisui-R (Rizo Tsukuba, Japan) or Arabidopsis using Plant RNA Reagent RNAzol (Life Technologies, Carlsbad, CA). Two micrograms of total RNA was treated with RNase-free DNaseI (Thermo Scientific, Waltham,

Histochemical GUS staining was performed according to the method described by Jefferson et al. (1987). Plants were incubated with GUS staining solution (50 mM HEPES-NaOH, 0.5 mM 5-bromo-4-chloro-3-indolylβ-D-glucuronide, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, and 0.05% [v/v] Triton X-100, pH 7.5) for 8 to 12 h at 37 °C. An ethanol wash was performed to stop the GUS reaction and remove chlorophyll from the plants. 2.6. qPCR All primers used for the Quantitative Real-Time PCR (qPCR) are listed in Supplemental Table S1. qPCR was performed with a LightCycler 96 System (Roche, Basel, Switzerland), using the FastStart Universal SYBR Green Master (ROX) (Roche, Basel, Switzerland). Quantities were determined from a standard curve and were normalized to the

Fig. 4. Nucleotide sequences of the 5′-flanking regions (from −991 to −1) of the IbRbcS1 gene determined in this study. Putative cis-acting elements including the TATA box, RbcS conserved, GATA motif, G-box, and I-box were masked with gray. Numbers are nucleotides relative to the 5′-flanking regions of the protein coding region.



A

respectively, were found in all introns in the IbRbcS1 and IbRbcS2 genes. The numbers of introns of RbcS genes were divided into three groups, the RbcS ORFs of which contained one (Oryza sativa and Zea mays), two (A. thaliana and Solanum lycopersicum), or three (S. lycopersicum RbcS2) introns, indicating that the exon/intron structure of the IbRbcS genes was more similar to those of the A. thaliana RbcS genes (Lebrun et al., 1987; Sugita et al., 1987; Krebbers et al., 1988; Xie and Wu, 1988). However, intron 2 (526 bp) of IbRbcS2 was longer than that of other plant RbcS genes. The deduced amino acid sequences of the IbRbcS1 and IbRbcS2 proteins contained 177 and 167 amino acid residues, respectively, and showed higher identities with other mature RbcS proteins (69–80% and 53–61%, respectively) (Fig. 2). The putative transit peptide of the IbRbcS1 protein was well conserved with other plant RbcS proteins, while that of the IbRbcS2 protein had a lower degree of identity.

IbRbcS1 pro:GUS

35S pro:GUS

4-1

5

6-3

3.2. Tissue-specific expression of RbcSs in sweet potato

B

IbRbcS1 pro:GUS 35S pro:GUS L

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4-1 L

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6-3 L

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GUS Actin8 Fig. 5. The expression of GUS in IbRbcS1 pro:GUS plants. (A) GUS histochemical staining of 2-week-old seedlings grown in vitro (CaMV 35S pro:GUS and IbRbcS1 pro:GUS plants). (B) Expression of GUS mRNA in the leaves and roots of transgenic Arabidopsis plants. CaMV 35S pro:GUS was used as a control. Amplified products were electrophoresed on 1.5% (w/v) agarose gels. L, leaves; R, roots.

amount of 18S rDNA. At least four experimental replicates were performed and standard errors were determined.

The tissue-specific expression levels of IbRbcS1 and IbRbcS2 mRNA in 2-month-old leaves, stems, and roots and 4-month-old tubers were examined by a semi-quantitative RT-PCR using IbRbcS1- or IbRbcS2specific primers. The expression of IbRbcS1 was detected in green tissues such as leaves and stems. The expression of IbRbcS2 was not detected in leaves, but was in stems, roots, and tubers (Fig. 3). Although number of RbcS genes in sweet potato was still unknown, these results implied that IbRbcS1 was one of the main RbcS family member in the leaves of sweet potato and also indicated that the promoter of IbRbcS1 was eligible for the green tissue expression of transgenes. Recently, a

A

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12 (h)

IbRbcS1 Actin

IbRbcS1 pro:GUS 4-1

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2.7. Accession number Nucleotide sequences of IbRbcS1 and IbRbcS2 have been deposited at DNA Data Bank of Japan (DDBJ: http://www.ddbj.nig.ac.jp). Accession number of IbRbcS1 and IbRbcS2 was LC036584 and LC036585, respectively.

0

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3

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GUS Actin8

3. Results and discussion

IbRbcS1 pro:GUS 6-3 3.1. Isolation and characterization of IbRbcSs In order to isolate sweet potato RbcS genes, genomic DNA was isolated from the leaves and subjected to genomic PCR using degenerated RbcS primers. Two different DNA fragments were amplified and sequenced at least three independent clones. Sequence analysis revealed that those DNA fragments were identified with similarity to the known RbcS genes of other plants. Based on the identified partial sequence of the RbsS genes, the remaining 5′ and 3′-sequences were obtained by cassette-ligation mediated PCR. We identified two IbRbcS genes (IbRbcS1 and IbRbcS2) and the IbRbcS cDNA fragments were then obtained by RT-PCR. A sequence analysis of these cDNA fragments revealed that three exons and two introns were assigned to the coding region in both IbRbcS genes (Fig. 1A and B). The consensus GT donor and AG acceptor sequences at the 5′- and 3′-termini of the introns,

0

3

6

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12 (h)

GUS Actin8 Fig. 6. Effects of light on the expression of the IbRbcS1 gene or GUS gene. (A) The expression levels of the IbRbcS1 gene in the leaves of sweet potato grown under a 14-h/10-h light/dark photoperiod. Actin was used as a control for the experiments. (B) The expression of the GUS gene was driven by the IbRbcS1 promoter in the leaves of transgenic plants. Actin8 was used as a control for the experiments. Amplified products were electrophoresed on 1.5% (w/v) agarose gels.


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functional RbcS that is not expressed in leaves was identified in rice (Morita et al., 2014). Detailed analyses of IbRbcS2 will be interesting for understanding the function of RbcS family in plants. We then cloned a −991 bp 5′ flanking sequence of the IbRbcS1 gene (Fig. 4). The putative cis-acting regulatory DNA elements in the 5′-flanking region of the IbRbcS1 gene were identified by the PLACE program (PLACE: http://www.dna.affrc.go.jp/PLACE/signalscan.html) (Higo et al., 1999). A TATA box element was located −86 to −80 from the ATG initiation site. The presumptive promoter sequence including AATCCAA, which was the same as the RbcS conserved sequence, was located at − 165 to − 159. The regulatory cis-acting sequences, the CCAAT element, GATA motif, G-box, and I-box, were present as observed in promoters from light-regulated genes including the RbcS genes of many plants. 3.3. Analysis of the IbRbcS1 promoter in Arabidopsis plants The sequences located upstream of the IbRbcS1 start codon were tested for their capacity to express the GUS reporter gene in the leaves of transgenic Arabidopsis plants. The − 991 bp putative IbRbcS1 promoter region was cloned in the binary vector pRI201-AN GUS (Takara, Japan) to drive β-glucuronidase. Histochemical staining revealed strong GUS signal in the leaves and stems of IbRbcS1 pro:GUS plants, in contrast to the constitutive GUS expression driven by the CaMV 35S promoter (Fig. 5A). To obtain further information on the expression patterns of the Ib rbcS1 promoter, semi-quantitative RT-PCR was performed using GUS-specific primers on total RNA extracted from leaves and roots. In accordance with GUS staining data, the semi-quantitative RT-PCR analysis revealed that the expression of GUS was only detected in the leaves of IbRbcS1 pro:GUS plants, but was detected in the leaves and roots of CaMV 35S pro:GUS plants (Fig. 5B). 3.4. The IbRbcS1 promoter conferred the light-response expression of transgenes We determined whether the expression of IbRbcS1 was regulated by light in the leaves of sweet potato by semi-quantitative RT-PCR using

4. Conclusion We herein obtained the complete cDNA sequences of RbcS genes from sweet potato (IbRbcS1 and IbRbcS2). The deduced amino acid sequences were well conserved with plant RbcS proteins. The expression of IbRbcS1 was green tissue-specific and was also regulated by light, whereas that of IbRbcS2 was specific to non-photosynthetic tissues, suggesting that the IbRbcS protein functioned as one of the main RbcS family member in sweet potato. The expression of GUS driven by the 5′flanking sequence of IbRbcS1 was green tissue-specific and was also regulated by light in transgenic plants. Compared with other plants, information regarding gene promoters in sweet potato is limited. The expression system using the IbRbcS1 promoter and its transit peptide sequences may offer promise as high expression of transgene in green tissue-specific manner in sweet potato. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.05.006.

1.2

1.0

Relative transcript levels

IbRbcS1-specific primers. Sweet potato plants were grown under a 14h/10-h light/dark photoperiod and samples were harvested at 3-h intervals from 0-h (dark period) to 12-h. The expression of IbRbcS1 was induced at 3-h and increased during the photoperiod (Fig. 6). We examined the expression of the GUS gene in Arabidopsis in order to evaluate the light-response capability of the IbRbcS1 promoter. In accordance with the expression pattern of IbRbcS1 in sweet potato, the expression of the GUS gene was induced at 3-h and increased during the photoperiod in IbRbcS1 pro:GUS plants (Fig. 6B). The expression of RbcS gene family members is known to be light-dependent, and a large number of light-responsive cis-elements have been identified in RbcS promoters. Two cis-acting elements, the I-box and G-box, were previously shown to be important for tissue-specific expression (Donald and Cashmore, 1990; Manzara et al., 1991). The expression of GUS driven by the tomato RbcS2 and RbcS3A promoters was detected in specific organs of the transgenic tomato such as the leaves and young tomato fruit (Iris et al., 1995). In these genes, the I-box and G-box were located − 600 to −100 bp upstream of the transcription initiation site. In the IbRbcS1 promoter region, the I-box and G-box were located −550 to −100 bp upstream of the start codon (Fig. 4), suggesting that these regulatory elements were involved in the light-responsive and green tissue-specific gene expression of the IbRbcS1 gene. In order to evaluate the activity of the IbRbcS1 promoter, we compared the expression levels of the GUS gene in the leaves of IbRbcS1 pro:GUS and CaMV 35S pro:GUS plants at 12-h light period after transition from the dark by qPCR. The qRT-PCR analysis revealed that the expression levels of the GUS gene in IbRbcS1 pro:GUS plants were same as those in CaMV 35S pro:GUS plants (Fig. 7). A previous study reported that the targeting of GUS into plastids using IbAGP1 transit peptides was highly effective for elevating the accumulation of foreign protein in transgenic plants (Kwak et al., 2007). Thus, it is likely that the application of IbRbcS1 transit peptide sequences for the accumulation of proteins in the chloroplasts of transgenic plants.

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0.4 Conflicts of interest Authors declare that there is no conflict of interest.

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Acknowledgments

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Control

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lbRbcS1 pro:GUS Fig. 7. Quantitative Real-Time PCR analysis of the GUS gene in transgenic Arabidopsis plants. CaMV 35S pro:GUS was used as a control. Values are the means ± SE of four replications for relative normalized expression.

We would like to thank Motoyasu Otani (Ishikawa Prefectural University) for providing sweet potato Kokei 14 plants, and Kumi Otori and Kota Kobayashi (Kinki University) for their technical advice on the experiments performed. This work was supported by the Core Research for Evolutional Science and Technology Project of the Japan Science and Technology Agency (Grant 2012-2017; to S. S.).



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A moso bamboo WRKY gene PeWRKY83 confers salinity tolerance in transgenic Arabidopsis plants.

Developmental and organ-specific changes in promoter DNA-protein interactions in the tomato rbcS gene family.

Identification of a 467 bp Promoter of Maize Phosphatidylinositol Synthase Gene (ZmPIS) Which Confers High-Level Gene Expression and Salinity or Osmotic Stress Inducibility in Transgenic Tobacco.

A nuclear gene encoding beta-amylase of sweet potato.

Pituitary-specific and hormonally regulated gene expression directed by the rat proopiomelanocortin promoter in transgenic mice.

Potato granule-bound starch synthase promoter-controlled GUS expression: regulation of expression after transient and stable transformation.

Ectopic expression of the floral homeotic gene AGAMOUS in transgenic Arabidopsis plants alters floral organ identity.

Analysis of the human tyrosine hydroxylase promoter-chloramphenicol acetyltransferase chimeric gene expression in transgenic mice.

Expression of the GUS-gene in the monocot tulip after introduction by particle bombardment and Agrobacterium.

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings.

Green fluorescent protein: an in vivo reporter of plant gene expression.